HIV infection of primary human cells typically requires interaction of Env gp120 with both the primary receptor CD4 and a chemokine receptor, either CCR5 or CXCR4, followed by Env gp41-induced fusion. Involvement of other factors and processes are likely, and their importance may differ between the principle target cells, macrophages (M?) and T-cells. Upon screening of a myeloid-specific monoclonal antibody (mAb) library, anti-CD63 mAb was found to block HIV entry into primary M?, but not T-cells. CD63 is a member of the tetraspanin membrane glycoprotein family, now implicated in HIV infection (MS #1), and other tetraspanins have been implicated in infection with HTLV-I (CD82), FIV (CD9) and Hepatitis C virus (CD81). Recent studies have shown inhibition with a GST fusion protein that includes the CD63 second extracellular loop (EC2), which further supports a specific role for CD63 in HIV infection. New data since the last submission also suggests a role for CD63 in endosomal processing of HIV, and so we will look at both early events, as well as events that occur later in the HIV life cycle, to more fully delineate the potential role of CD63 in HIV infection. These studies may lead to development of novel antiretroviral therapies. We will pursue these aims over five years: Aim 1. To test the hypothesis that CD63 is involved with early HIV replication events, we will investigate virus uptake and initiation of reverse transcription in M? and T cells/cell lines. Aim 2. To test the hypothesis that CD63 is generally required for infection of primary M?, as well at T cells/cell lines, we will assess trafficking and assembly of viral proteins, particularly Gag (and also Env) with and without CD63 silencing with small inhibiting RNAs (siRNA). We will also silence varions Rab proteins, which are cellular GTPases that traffic cellular (and viral) proteins through endosomal pathways in order to identify steps of HIV trafficking affected by CD63. Aim 3. To test the hypothesis that CD63 plays a role in infection of M? (and other cells) by diverse primary HIV 1 strains, we will assess anti-CD63 and CD63-EC2 susceptibility of both subtype B and non-subtype B strains, including those that use CCR5 exclusively (R5, CXCR4 exclusively (X4) or dual-tropic strains (R5X4).